Prevalence of Primary Resistance Mutations to Integrase Inhibitors in ...

Prevalence of Primary Resistance Mutations to Integrase Inhibitors in Treatment-Naïve and -Experienced Patients Infected With B and Non-B HIV-1 Variants Carolina Gutiérrez,1 Beatriz Hernández-Novoa,1 María Jesús Pérez-Elías,1 Ana María Moreno,1 África Holguín,2 Fernando Dronda,1 José Luis Casado,1 and Santiago Moreno1 1 Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid Spain; 2HIV-1 Molecular Epidemiology Laboratory, Microbiology Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

Background: Raltegravir (RAL) constitutes the first available integrase strand transfer inhibitor (INSTI) available in clinical practice. Three independent pathways have been described to confer resistance to RAL. Secondary mutations with little effect on INSTI susceptibility and additional substitutions with an uncertain role have also been described especially in HIV-1 non-B variants. Methods: We evaluated the prevalence of primary, secondary, and additional resistance mutations to INSTIs in patients naïve to RAL or elvitegravir (EGV) carrying different HIV-1 variants. Results: A total of 83 patients infected by B HIV-1 subtype (64%) or non-B HIV-1 variants (36%) were evaluated. No primary mutations to RAL or EGV were found in the integrase sequences analyzed. Secondary mutations were detected in only 5 patients. Additional mutations were found in both in B and non-B variants. According to the geno2pheno algorithm, some of the secondary mutations detected (L74V, E138K, G163RS, and V151I) have been associated with a reduced estimated susceptibility to RAL and only the E138K mutation has been associated with a decreased estimated susceptibility to EGV. No virological failure was observed after RAL was administrated in 17 patients carrying 1 or more additional substitutions in the absence of primary or secondary mutations. Conclusions: No primary resistance mutations to INSTI were found in treatment-naïve or -experienced patients infected with B or non-B HIV-1 variants. The vast majority had some polymorphic and nonpolymorphic substitutions; however response to RAL was excellent in patients who harbored one or more of these mutations. We could not identify any clinical factors associated with the presence of any of these mutations. Key words: mutations, non-B variants, raltegravir, subtype B

ntegrase mediates the insertion or integration of the HIV-1 DNA into the host genome.1 In the process of viral infection, inhibition of integration results in an irreversible block of HIV-1 replication, with most of the unintegrated viral DNA being degraded by a variety of cellular enzymes. Integrase inhibitors target the strand transfer reaction, 1 of the 3 steps of the integration process; for that reason, they are commonly named more specifically as integrase strand transfer inhibitors (INSTIs).2 Raltegravir (RAL) constitutes the first available INSTI in clinical practice and has shown potent and durable antiretroviral activity in naïve and pretreated HIV-1–infected patients.3-5 In addition to wild-type virus, RAL has shown significant activity against HIV-1 strains resistant to reverse

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transcriptase, protease, and entry inhibitors.6,7 However, in conditions of incomplete viral suppression, specific mutations could arise and confer resistance to RAL. Elvitegravir (EGV) is another INSTI in the last phases of clinical development that shows some degree of cross resistance with RAL.8,9 Address for correspondence: Carolina Gutiérrez, PhD, Department of Infectious Diseases, Hospital Ramón y Cajal, Carretera de Colmenar, Km 9,100, 28034 Madrid, Spain; phone: +34-91-3368711; fax: +34-91-336-8792; e-mail: cgutierrezm.hrc@salud. madrid.org HIV Clin Trials 2013;14(1):10–16 © 2013 Thomas Land Publishers, Inc. www.thomasland.com doi: 10.1310/hct1401-10

PRIMARY RESISTANCE MUTATIONS TO INTEGRASE •

Three independent pathways mediated by 3 different mutations (Q148R/K/H, N155H, and Y143R/C) have been described to confer resistance to RAL.10,11 Secondary mutations have also been identified that have little effect on RAL susceptibility but that can contribute to increased RAL resistance when present together with 1 or more primary mutations.12-14 Other additional substitutions, divided into nonpolymorphic (T125K, A128T, Q146K, N155S, K160D) and polymorphic (V72I, A154I, V165I, V201I), can be detected naturally in the integrase gene, especially in patients with non-B HIV-1 variants, with an uncertain role on the HIV-1 susceptibility to INSTIs.15,16 The genetic barrier of INSTIs is lower than that of protease inhibitors (PIs) and the majority of nucleoside reverse transcriptase inhibitors (NRTIs). The development of resistance mutations occurs more rapidly under RAL pressure than with PIs and NRTIs and is usually detected in the first several months of therapy.17 With the increasing use of RAL in both treatment-naïve and -experienced patients, the possibility of transmitted resistance is of concern, as is the potential decreased susceptibility to INSTIs in patients with multiple failures to other drugs. Some studies have failed to detect primary resistance mutations in RAL-naïve patients,14,18 but little is known about the distribution of resistance mutations according to HIV-1 subtypes as well as the frequency of secondary mutations and their impact on clinical response.10,19,20 The aim of this study was to evaluate the prevalence of primary, secondary, and additional resistance mutations to INSTIs, and their clinical implications in patients who had never been treated with INSTIs, according to HIV-1 subtype (including HIV-1 subtype B and non-B variants).

METHODS Patients We performed a retrospective analysis in stored plasma samples of HIV-1–infected patients followed at 2 Infectious Diseases Departments (Hospital Ramón y Cajal, Madrid, and Hospital Dr. Negrín, Las Palmas, Spain) from 2007 to 2009. No patient had been previously treated with RAL. All eligible patients had a viral load greater than 1,000 copies/mL as determined by Versant HIV-1 RNA 3.0 Assay (bDNA) (Siemens Healthcare

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Diagnostics, Tarrytown, New York, USA). For purposes of analysis, they were grouped according to previous antiretroviral treatment and subtype. Methodology For detection of integrase resistance mutations, 1 mL of cryopreserved plasma sample was concentrated by centrifugation during 2 hours at 32,000 rpm and 4°C. The obtained pellet was used for RNA extraction using the QIAamp viral kit (Qiagen, Valencia, California, USA) according to the manufacture’s recommendations. A polymerase chain reaction (PCR) amplified product of 864 base pairs (bp) corresponding to the HIV-1 integrase gene was obtained using a commercially available kit (Viroseq HIV-1 Integrase Genotyping Kit; Celera Diagnostic, Abbot Molecular, Alameda, California, USA). This kit is designed to detect resistance mutations in the integrase region using a single-step reverse transcription-PCR (RT-PCR) with the following amplification conditions: prePCR holds [20oC for 10 min; 55oC for 30 min; 95oC for 15 min] – 45 cycles x [94oC for 15 s, 55o C for 30 s, 68o C for 1 min and 30 s] – 68oC for 7 min. PCR products were purified using spin columns (QIAquick PCR Purification Kit, Qiagen). DNA sequencing reaction was performed using premixed BigDye terminator (Applied Biosystems, Foster City, California, USA) with sequence reagents and appropriate primers. Conditions for cycle sequencing were 25 cycles x [96oC for 10 s, 50oC for 5 s, and 60oC for 4 min]. An ABI PRISM 310 Genetic analyzer (Applied Biosystems, Carlsbad, California, USA) was used to sequence products. Integrase sequences were edited using the SeqMan Software version 5.0.1 (DNAStar, Madison, Wisconsin, USA). Resistance to INSTIs was estimated on the basis of the geno2pheno report (Integrase Resistance Prediction, v. 1.0; www.geno2pheno.org) Mutations in retrotranscriptase (RT) and protease (PR) genes were determined using the HIV genotyping system (Celera Diagnostics Viroseq HIV-1 Genotyping System, Abbot Molecular) according to the manufacturer’s recommendations. RNA extraction was performed using the QIAamp viral kit (Qiagen), followed by a reverse transcription (65oC for 30 s, 42oC for 65 min, 99oC for 5 min) and by a single 40-cycle PCR (50oC for 10 min, 93oC for 12 min, 93oC for 20 s, 64oC for 45 s, 66oC for 3 min, 72oC for 10 min) with the

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HIV CLINICAL TRIALS •

appropriate primers in each PCR. The DNA PCR product of 1.8 kb obtained was purified using spin columns QIAquick PCR Purification Kit (Qiagen). DNA sequencing reaction was performed using premixed BigDye terminator (Applied Biosystems) with sequencing reagents and appropriate primers. The conditions for cycle sequencing were the same as those used for integrase sequencing described previously. Sequence data were analyzed using the Celera Diagnostics ViroSeq HIV-1 Genotyping System software that permitted the alignment of full-length sequences. Antiretroviral resistance was estimated on the basis of the Stanford HIV drug resistance database report (www.hivd.stanford. edu). The subtype was determined according to the sequences from the pol region. Statistical Analysis All statistical analyses were performed using IBM SPSS software v.15 (IBM, Armonk, New York, USA). The results were expressed as percentage, median, and interquartile range (IQR).

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Table 1. Baseline characteristics of the patients with integrase sequence analyzed (N = 83) Variable

n (%)a

Male HIV-1 variants Subtype B Subtypes non-B and recombinants Median HIV-1 RNA load, copies/ mL (IQR) Median CD4 count, cells/mm3 (IQR) Treatment-naïve Treatment-experienced Previous NRTI Previous NNRTI Previous PI Resistance mutations in treatment-experienced patients NRTI NNRTI PI

57 (69) 53 (64) 30 (36) 19,400 (4,120-79,816) 455 (313-576) 64 (77.1) 19 (22.9) 100% 95% 89.5%

32% 42% 42%

Note: NNRTI = non-nucleoside reverse transcriptase inhibitors; NRTI = nucleoside reverse transcriptase inhibitors; PI = protease inhibitors. a Values are given as n (%) unless otherwise indicated.

RESULTS A total of 109 patients (69 with HIV-1 subtype B and 40 carrying non-B HIV-1 variants) had available samples for analysis. Among them, 83 patients (76%) had an HIV-1 RNA load greater than 1,000 copies/mL. Integrase sequences could be obtained in all of them: 53 with subtype B and 30 harboring non-B variants recombinant CRF02_AG [18 patients], CRF09_cpx [3], subtype A [3], CRF06_cpx [2], CRF12_BF [2], CRF14_BG [1], and subtype G [1]). Most (93%) of the 30 patients infected with non-B HIV-1 variants were naïve to antiretroviral therapy, compared to 36 patients (68%) infected with B subtypes. Among the 83 patients, 69% were male, with a median HIV-1 RNA load of 19,400 copies/mL (IQR, 4,120-79,816) and median CD4 count of 455 cells/mm3 (IQR, 313-576). Drug-experienced patients had received NRTI (100%), non-nucleoside reverse transcriptase inhibitors (NNRTI; 95%), and PI (89.5%). Resistance mutations to NRTI, NNRTI, and PI were found in 32%, 42%, and 42% of patients, respectively (Table 1). Primary and Secondary Mutations No principal mutations to RAL or EGV resistance were found in the whole integrase sequences

analyzed. Secondary mutations could be detected in only 5 patients, all of whom were naïve to antiretroviral drugs. In the B subtype group, 3 patients showed the mutations E138K and/or V151I; in the non-B HIV-1 variants, they showed the mutations G163RS and/or L74M (Table 2). The impact on phenotypic resistance was estimated with the geno2pheno algorithm. None of the mutations detected have been associated with a reduction in RAL susceptibility. Only the E138K mutation, detected in 1 patient, has been associated with a decreased susceptibility to EGV. Additional Mutations Additional mutations were frequently found in B and non-B variants. Overall, V201I (54 patients, 65%) and V72I (53 patients, 64%) were the most prevalent substitutions detected. Other previously described nonpolymorphic mutations (T125K, A128T, Q146K, N155S, and K160D) could not be found in any of the 83 samples analyzed. Most patients infected with non-B HIV-1 variants (93%) presented 1 or more of these mutations, the most frequent being V201I (86.7%) and V72I (67.8%). The 2 mutations were found together in


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Table 2. Distribution of integrase mutations in the in treatment-naïve and -experienced patients infected with B and non-B HIV-1 Treatment-naïve Subtype B (n = 36)

Non-B variants (n = 28)

Treatmentexperienceda (n = 19)

0

0

0

0

–

With secondary mutations L74M E138K V151I G163RS

0 1 2 0

1 0 0 1

0

1 1 2 1

RAL RAL, EGV RAL RAL

With additional mutations V72I S119R V165I V201I

22 3 1 20

18 0 3 24

13 0 0 10

53 3 4 54

– EGV – –

With primary mutationsc

Total (N = 83)

INSTIs with reduced estimated susceptibilityb

Note: EGV = elvitegravir; INSTIs = integrase strand transfer inhibitor; RAL = raltegravir. a Treatment-experienced patients were infected with B subtype HIV (n = 17) and non-B subtype HIV (n = 2). b According to the geno2pheno algorithm. c Primary mutations analyzed include N155H, Q148K/R/H, and Y143R/C.

57% of patients. No association was found between the presence of these mutations and HIV viral load in plasma or other patient characteristics (data not shown). No impact of the mutations on the estimated susceptibility to integrase inhibitors was found, including in the 2 patients of this group who had secondary mutations. The majority of patients (81%) with B HIV-1 subtype showed some additional mutations as well. The most prevalent substitutions were V72I (64%) and V201I (53%). The S119R mutation, associated with a partial reduction in the susceptibility to EGV according to the geno2pheno estimation, was detected in 3 patients. One additional patient had the V165I mutation in combination with V72I. Again, no association was found between the presence of these mutations in HIV-1 subtype B and plasma HIV-1 RNA viral load, CD4 count, or the detection of mutations in the RT or in the PR genes.

included RAL. Accompanying drugs included 2 NRTIs plus either ritonavir-boosted darunavir (50% of the patients), maraviroc (33%), or etravirine (17%). In 3 cases, 2 of these drugs were included in the regimen. The median HIV-1 RNA load at the time of receiving RAL was 7,943 copies/mL (IQR, 1,000-81,548) and the median CD4 count was 322 cells/mm3 (IQR, 199-449). None of the participants had primary or secondary mutations to INSTI, although 88% showed 1 or more of the additional mutations in the integrase sequence (V72I [70.6%], V201I [52.9%], and S119R [11.8%]). As a result of multiple previous therapeutic failures, all of them presented main resistance mutations associated to NRTI (40%), NNRTI (53%), and PI (53%). No virological failure was observed after administration of RAL-containing regimens. All 17 patients achieved HIV-1 RNA load below 50 copies/mL, with a median increase in the CD4 count up to 413 cells/mm3 (IQR, 237-578).

Response to RAL Therapy Of the 83 patients included in the analysis, 17 patients who were HIV-1 subtype B–infected and had multiple previous antiretroviral treatment failures received treatment with a regimen that

DISCUSSION We could not detect known primary resistance mutations to integrase inhibitors in treatmentnaïve or -experienced patients infected with

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subtype B or non-B HIV-1 variants who were never exposed to INSTI. In contrast, secondary mutations were found in a limited number of patients, and the vast majority had some polymorphic and nonpolymorphic substitutions. The impact on the estimated phenotypic susceptibility to first-generation integrase inhibitors is very limited; indeed, response to RAL-containing regimens was excellent in patients who harbored 1 or more of these mutations. The lack of primary resistance to INSTI has been previously reported, especially among the B HIV-1 subtype.15,21,22 The first case of transmitted INSTIs was detected 3 years after RAL was approved by the US Food and Drug Administration.23,24 Most studies were performed at a time when RAL was not extensively used. Thus, there was subsequently little risk of transmitted resistance, which explains the findings. However, it is reassuring to note that significant resistance does not occur naturally in patients who are not exposed to antiretroviral drugs or in heavily treated patients who have received multiple antiretroviral regimens with resistant viruses to other drug families. According to our results and to previous reports, substitutions in the RT and in the PR are not associated with changes in the integrase.6,7,16,25 Moreover, a recent report failed to detect primary resistance to INSTI in recently infected patients who acquired infection in years in which RAL was already in the market.26 Less information has been reported on the presence of primary integrase mutations in HIV-1 non-B variants.22,27-30 These variants are responsible for 90% of the 34 million infections worldwide.31 In Spain, non-B variants are increasing among newly diagnosed HIV-1 native Spaniards and immigrants.32 They represent 12.2% of infections in naïve subjects, which are mainly caused by recombinants.33 The HIV-1 integrase region is highly conserved among HIV-1 subtypes.34 As some in vitro studies show,35 HIV-1 integrase inhibitor would be active in all HIV-1 variants. In addition, clinical studies have confirmed these previous in vitro results showing similar mutation patterns of RAL resistance in HIV-1 subtype B and non-B HIV-1 variants.29 However, the fact that some polymorphisms are more frequent in these non-B variants22,36 could lead to the assumption that primary or secondary mutations should be more frequent as well. Our study confirms the absence of primary mutations, although our conclusions may be

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limited to naïve patients since only 2 patients who were treatment-experienced and infected with a non-B HIV-1 variant could be evaluated. A high proportion of patients with B and non-B HIV-1 variants showed some additional substitutions selected by investigational INSTI, although these have not been described to be associated with RAL or EGV resistance.14,15,37 The nonpolymorphic mutation N155S previously selected in vitro could have a remarkable role in showing a decrease in both RAL and EGV susceptibility.9,38 Some authors have found that these changes are related to increased levels of resistance to INSTI in the presence of primary mutations.39,40 In 1 study, 1 or more of these mutations in the integrase gene (especially V72I and/or V201I) were found at baseline in 9 of 11 patients failing an RAL-containing regimen. However, they could not establish any relationship between the presence of these polymorphic substitutions at baseline and the subsequent selection of the different pathways of INSTI resistance.39 We could not identify factors that were associated with a higher prevalence of primary substitutions in the integrase region, including viral HIV-1 subtype, plasma HIV-1 RNA load, CD4 cell count, previous antiretroviral treatment, or the number of mutations in the PR or the RT. It must be acknowledged, however, that the large percentage of patients showing substitutions precludes us from obtaining significant associations. Among the patients included in this study, we could evaluate the impact of polymorphic substitutions in the virological efficacy of RAL in 17 patients from the B HIV-1 subtype group. They had a previous history of antiretroviral treatment; had developed resistance mutations to NRTIs, NNRTIs, and PIs; and received an RAL-containing regimen as salvage therapy. Despite the fact that 82% showed additional substitutions in the integrase gene, including the S119R substitution related to a partial reduction in the susceptibility to EGV, no virological failures were detected. This could suggest that the high prevalence of substitutions in the integrase detected in this study does not interfere with the efficacy of INSTI, as previous studies supported.22,29,41 These drugs can be safely administered to patients who have not failed previously, as shown in clinical trials of initial or salvage therapy.7,10,25,42-44 It could be argued that all the patients received regimens that included multiple drugs, so the potential loss of activity of RAL could


be compensated by other active drugs in the combination. The efficacy of RAL in combination with only 2 nucleosides in patients infected with HIV-1 harboring secondary or additional resistance mutations to INSTI remains to be established. In summary, we did not detect the presence of primary resistance to INSTIs in HIV-1 subtype B and non-B variants. The presence of secondary mutations was limited to a few patients without a reduction in RAL or EGV susceptibility. Although the presence of polymorphic and nonpolymorphic substitutions was detected in the majority of the evaluated patients, no impact on RAL efficacy was found in the patients treated with RAL. More studies are necessary to evaluate the impact of secondary and additional substitutions in patients receiving new integrase inhibitors in development. ACKNOWLEDGMENTS Financial support: This research was supported in part by the Spanish AIDS Network “Red Temática Cooperativa de Investigación en SIDA (RD06/0006)” and the Mutua Madrileña Project FM 2008/145. Additional contributions: We thank Carmen Page and María Pumares for excellent technical and coordinator assistance, and Dr. María José Pena from “Dr. Negrín Hospital”, Las Palmas, Spain, for the cession of non-B variants samples. REFERENCES 1. Chiu TK, Davies DR. Structure and function of HIV-1 integrase. Curr Top Med Chem. 2004;4:965–977. 2. Hazuda DJ, Felock P, Witmer M, et al. Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells. Science. 2000;287:646–650. 3. Markowitz M, Morales-Ramirez JO, Nguyen BY, et al. Antiretroviral activity, pharmacokinetics, and tolerability of MK-0518, a novel inhibitor of HIV-1 integrase, dosed as monotherapy for 10 days in treatment-naive HIV1-infected individuals. J Acquir Immune Defic Syndr. 2006;43:509–515. 4. Markowitz M, Nguyen BY, Gotuzzo E, et al. Rapid and durable antiretroviral effect of the HIV-1 integrase inhibitor raltegravir as part of combination therapy in treatment-naive patients with HIV-1 infection: results of a 48-week controlled study. J Acquir Immune Defic Syndr. 2007;46:125–133. 5. Markowitz M, Nguyen BY, Gotuzzo E, et al. Sustained antiretroviral effect of raltegravir after 96 weeks of combination therapy in treatment-naive patients with HIV-1 infection. J Acquir Immune Defic Syndr. 2009;52:350–356.

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